| Background: Post-stroke cognitive impairment(PSCI)refers to the decline in cognitive function that occurs after stroke,and is the most common functional impairment after stroke,seriously affecting patients’ quality of life and survival time.However,the diagnosis rate of PSCI is low in clinical practice,and there are relatively few relevant studies and controversial clinical prevention and treatment protocols.Research on the diagnosis,pathological mechanism and early intervention of PSCI has become a hot topic of current research.Acupuncture is an important tool in Chinese medicine for the treatment of diseases such as dementia and amnesia,and it is also widely used in the treatment of post-stroke complications with definite efficacy.A generalization of basic research shows that cognitive-related behavioral studies require attention to the following aspects: first,according to the clinical incidence of PSCI,the incidence of PSCI in stroke model animals is not 100%,and the subjects need to be screened more strictly;second,the higher incidence and longer duration of motor dysfunction in rats/mice after stroke can interfere with cognitive function-related behavioral experiments.Third,most of the studies used to assess cognitive function only used the Morris water maze,which is relatively single and failed to reasonably and effectively evaluate other cognitive impairments besides spatial memory;fourth,the effect of transient cognitive impairment in rats/mice during the acute phase of stroke on the experimental effect needs to be excluded;fifth,the study mechanism mainly focused on the pathological process of stroke leading to PSCI Fifth,the study mechanism is mainly focused on the pathological process of PSCI caused by stroke,and the direct effect of acupuncture points is lacking.In this study,we proposed to establish an animal model of ischemic stroke by MCAO,screen mice with post-stroke cognitive dysfunction without voluntary motor dysfunction by using absentee field experiment,novelty recognition experiment,Barnes’ maze and Morris’ water maze experiment,give them electroacupuncture stimulation,then evaluate their cognitive function improvement by cognitive behavioral experiment,and explore the mechanism of action from histopathological and molecular biological perspectives.The mechanism of action was investigated from histopathological and molecular biological perspectives.Objective: To study the improvement effect and pathological mechanism of acupuncture at "Baihui,Yintang,and Shuigou" points on Post-stroke cognitive impairment(PSCI)in mice.Method: 52 SPF male C57BL/6 mice were used to establish a MACO model using the middle cerebral artery occlusion method(N=38).In the Sham group,only cervical blood vessels were isolated without insertion of a tether(N=14).After surgery,mice were routinely reared.After 28 days,open field experiments,new object recognition experiments,Barnes maze,and Morris water maze experiments were used to evaluate their motor and cognitive functions.Based on the level of cognitive function in Sham group,the MCAO group was divided into three groups: non cognitive impairment group(MCAO non PSCI,MNP,N=14),post stroke cognitive impairment group(MCAO PSCI,MP,N=13),and electroacupuncture intervention group(MCAO PSCI-EA,MPEA,N=11).In the MPEA group,electroacupuncture was performed at "Baihui,Yintang" acupoints,with a density wave of 2 Hz,1 m A,20 minutes,then supplemented by pricking at "Shuigou" acupoints.The mice in the other groups only grabbed and fixed daily,without any intervention.After treatment,the cognitive function of mice in each group was tested again using open field experiments,new object recognition experiments,Barnes maze,and Morris water maze experiments.Neuron status of hippocampal region was observed using Nissl’s staining,and the expression of synaptic associated proteins Glu A1,Synaptophysin(Syp),and Synaptomin-1(Syt-1)in the hippocampus was detected using Western blot and immunohistochemical staining(IHC).Results: 1.To exclude the influence of the impairment of voluntary motor function on the assessment of cognitive function after MCAO,the open field test was performed at 28 d after operation to observe the motor ability of mice in the autonomous activity state,and it was found that the motor distance and motor speed of mice in the open field test after MCAO were not significantly different from those in the Sham(P < 0.05).2.To clarify the cognitive impairment after MCAO,the Barnes maze and Morris water maze experiments were used to evaluate.In the Barnes experiment,the time required to find the target area and the distance traveled were significantly higher in the MCAO than in the Sham (P < 0.01),and the frequency of exploration,the time spent in the target area and the distance moved in the target area were significantly less than in the Sham(P <0.05).In the Morris water maze experiment,the exploration time and distance travelled in the MCAO were significantly longer than those in the Sham(P < 0.01),and in the spatial exploration experiment,the number of times the MCAO crossed the original platform was significantly lower than that of the Sham(P < 0.05).The these results indicate the cognitive impairment of MCAO mice at 28 d postoperatively.Further analysis revealed that the incidence of PSCI was 58.33% when mice were randomly divided into post-stroke no cognitive impairment(MCAOnon-PSCI,MNP)(N=14),and post-stroke cognitive impairment(MCAO-PSCI)(N=24),using exceeding the mean value of Morris water maze escape latency in Sham as the baseline.The mice with PSCI were divided into electroacupuncture intervention group(MCAO-PSCI-EA,MPEA)(N=11)and no electroacupuncture stimulation group(MCAO-PSCI,MP)(N=13)into the follow-up experiments.3.In order to clarify the effect of acupuncture on the spontaneous motor ability of MCAO mice,in the open field experiment,there was no difference in the total distance traveled by the mice in each group;compared with the Sham,the number of times the mice explored the central zone and the activity time in the central zone were significantly reduced in the MP and MPEA groups(P < 0.05),however,there was no difference between the two groups(P > 0.05).Suggesting that acupuncture has no effect on the motor function of PSCI mice,but acupuncture possibly causes emotional impairment in mice.4.To clarify the effects of acupuncture on the cognitive function of MCAO mice,novel object recognition and Morris water maze were used for evaluation.In the Barnes maze experiment,the time required to find the platform in the MP group was significantly longer than that in the Sham and MNP(P < 0.05),while it was not statistically different from that in the MPEA group.In the Morris water maze,the evasion latency was significantly longer in the MP than in the MPEA(P < 0.05);in the water maze spatial exploration experiment,the time taken to find the original platform was significantly delayed in the MP group compared with the MPEA(P < 0.05).5.In order to clarify the histopathological changes of brain after electroacupuncture treatment,the brain tissues of mice were observed by Nissl staining.The results showed that in the affected hippocampal region,large infarct foci were seen in the MP and MPEA,and observation of the contralateral hippocampal regions revealed that the number of normal neurons in CA1 and CA2 regions was less in the MP than in the MPEA(P < 0.05);IHC detection revealed that the expression of Syp in the contralateral hippocampal region of mice in the MPEA was significantly higher than that in the MP(P < 0.05).It indicates that electroacupuncture intervention can improve the structural destruction of ischemic brain tissue.6.Western blot examination of the affected hippocampus revealed that the expression of Glu A1,Syp,and Syt-1,proteins related to synaptic signaling,was significantly increased in the hippocampal region of mice in the MPEA compared with the MP(P < 0.05).This indicates that electroacupuncture intervention can improve synaptic structural plasticity in ischemic brain tissue.Conclusion: Acupuncture points of Baihui,Yin Tang and Shuigou significantly improved cognitive dysfunction in MCAO mice,which may be related to the regulation of synapticrelated protein expression in hippocampal neurons and reduction of synaptic damage and hippocampal neuronal damage. |
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